Acceptability of Iron- and Zinc-Biofortified Pearl Millet (ICTP-8203)-Based Complementary Foods among Children in an Urban Slum of Mumbai, India

Biofortification, a method for increasing micronutrient content of staple crops, is a promising strategy for combating major global health problems, such as iron and zinc deficiency. We examined the acceptability of recipes prepared using iron- and zinc-biofortified pearl millet (FeZnPM) (~80 ppm Fe, ~34 ppm Zn, varietal ICTP-8203), compared to conventional pearl millet (CPM) (~20 ppm Fe, ~19 ppm Zn) in preparation for an efficacy trial. Our objective was to examine the acceptability of FeZnPM compared to CPM among young children and mothers living in the urban slums of Mumbai. Standardized traditional feeding program recipes (n = 18) were prepared with either FeZnPM or CPM flour. The weight (g) of each food product was measured before and after consumption by children (n = 125) and the average grams consumed over a 3-day period were recorded. Mothers (n = 60) rated recipes using a 9-point hedonic scale. Mean intakes and hedonic scores of each food product were compared using t-tests across the two types of pearl millet. There were no statistically significant differences in consumption by children (FeZnPM: 25.27 ± 13.0 g; CPM: 21.72 ± 6.90 g) across the food products (P = 0.28). Overall mean hedonic scores for all recipes were between 7 to 9 points. CPM products were rated higher overall (8.22 ± 0.28) compared to FeZnPM products (7.95 ± 0.35) (P = 0.01). FeZnPM and CPM were similarly consumed and had high hedonic scores, demonstrating high acceptability in this population. These results support using these varieties of pearl millet in a proposed trial [http://Clinicaltrials.gov ID: NCT02233764; Clinical Trials Registry of India (CTRI), reference number REF/2014/10/007731, CTRI number CTRI/2015/11/006376] testing the efficacy of FeZnPM for improving iron status and growth.

Biofortification, a method for increasing micronutrient content of staple crops, is a promising strategy for combating major global health problems, such as iron and zinc deficiency. We examined the acceptability of recipes prepared using iron-and zinc-biofortified pearl millet (FeZnPM) (~80 ppm Fe, ~34 ppm Zn, varietal ICTP-8203), compared to conventional pearl millet (CPM) (~20 ppm Fe, ~19 ppm Zn) in preparation for an efficacy trial. Our objective was to examine the acceptability of FeZnPM compared to CPM among young children and mothers living in the urban slums of Mumbai. Standardized traditional feeding program recipes (n = 18) were prepared with either FeZnPM or CPM flour. The weight (g) of each food product was measured before and after consumption by children (n = 125) and the average grams consumed over a 3-day period were recorded. Mothers (n = 60) rated recipes using a 9-point hedonic scale. Mean intakes and hedonic scores of each food product were compared using t-tests across the two types of pearl millet. There were no statistically significant differences in consumption by children (FeZnPM: 25.27 ± 13.0 g; CPM: 21.72 ± 6.90 g) across the food products (P = 0.28). Overall mean hedonic scores for all recipes were between 7 to 9 points. CPM products were rated higher overall (8.22 ± 0.28) compared to FeZnPM products (7.95 ± 0.35) (P = 0.01). FeZnPM and CPM were similarly consumed and had high hedonic scores, demonstrating high acceptability in this population. These results support using these varieties of pearl millet in a proposed trial inTrODUcTiOn Iron deficiency is the most common micronutrient deficiency in the world and particularly impacts child health in resource-limited settings such as urban slums in India (1). With or without anemia, low iron status impairs growth (2), immune (3), and cognitive functions (4) in infants and young children. Preliminary data from children in our study population in Mumbai showed that iron intake was 35% that of the Indian RDA (5,6) and that nearly 60% of children were iron deficient (serum ferritin < 12 ng/mL) (7). Zinc deficiency affects nearly 2 billion people worldwide and is associated with short stature, delayed development, and increased morbidity particularly from diarrhea (8). Collectively, these deficits, particularly during the first 1,000 days of life, result in long-term health consequences such as poor school performance and decreased work capacity (9). Biofortification of staple crops with iron is a sustainable agricultural approach that can help prevent and treat iron deficiency in vulnerable populations (10). In the process of biofortification, the concentration and bioavailability of essential micronutrients in staple food crops is increased by traditional plant breeding techniques (11,12). Pearl millet (Pennisetum glaucum, and locally known as bajra in Hindi) is a nutritious, hardy, drought-tolerant grain that is a staple of the traditional diet in many areas of India including Maharashtra, Gujarat, and Rajasthan, where per-capita consumption is 80-100 g (10,13).
A locally grown variety of iron-and zinc-biofortified pearl millet (ICTP-8203 or FeZnPM) with four times higher iron concentration than conventional pearl millet (CPM) has the potential to improve iron status in these populations (10,14). Traditionally, pearl millet is ground into flour, roasted, and consumed in the form of non-leavened breads called bhakri or roti as part of the daily diet. ICTP-8203, shown to have comparable if not higher yield than conventional varieties (15), has demonstrated efficacy in improving iron status in older children who consumed bhakri twice daily for 6 months (10,16). However, flatbreads like bhakri are not ideal weaning or complementary foods for young children and infants to consume due to their tougher and chewier texture. The main objective of this study was to formulate and test the acceptability (in terms of volume consumed and sensory characteristics) of new pearl millet-based palatable complementary food products for weaning infants. The food products with highest acceptability would be ideal candidates for a randomized controlled trial testing the efficacy of biofortified pearl millet for improving iron status in infants and young children.

MaTerials anD MeThODs intervention
Pearl millets CPM and ICTP-8203 were transported from Maharashtra and Gujarat, respectively, in 50-kg gunny bags and stored in a climate-controlled space (humidity level < 50%, 25°C). Pearl millet was transferred from the gunny bags into steel canisters and transported to SNDT Women's University, Mumbai, India, for storage and preparation. The concentrations of iron, zinc, and aluminum in whole grain were quantified after nitric acid digestion using inductively coupled plasma optical emission spectrometry (ICP-OES) at the International Crops Research Institute for the Semi-Arid Tropics research laboratory in Telangana, India. Confirmatory testing in the USDA-ARS lab at Cornell University (courtesy, Dr. Ray Glahn), was used to test for iron and zinc concentrations, along with assessment of potential contamination via aluminum concentrations using ICP-OES.

recipe Formulation and Production
Ingredients and formulations were based on traditional weaning and complementary foods in this region. The teaching food laboratory at SNDT Women's University was utilized for recipe development. The recipes were prepared by Master of Science in Food, Science, and Nutrition students and staff from the department of Food Science and Nutrition under the guidance of the study investigators and community coordinator. Standard hygiene and sanitation procedures were maintained throughout.

study setting
The study site was a feeding center within a large slum known locally as Nehru Nagar, in Vile Parle, a suburb in Mumbai. The feeding center was a room generally used for Integrated Child Development Services activities (17). This room was equipped with electricity and fans.

study Design and Protocol
This study was conducted from January to December 2015. Mothers were given an information sheet describing the pearl millet crop and its potential health benefits. Community health workers explained to mothers that they would be given foods prepared using both high-iron/zinc and conventional pearl millets to consume.
Based on preliminary data indicating preferred times of day for feeding, mothers were requested to come to the feeding center between 11:00 a.m. and 5:00 p.m. each day to maximize participation. If a child refused food or if mothers needed to leave before the child finished, mothers were requested to return to the center to continue feeding. Mothers were also encouraged to come between breast-feeding periods to encourage consumption of the complementary food. Participants consumed each food product over a 3-day period to allow the child to become familiar with any potential new tastes or flavors. First, all CPM products were tested for acceptability (days 1-3), followed by all FeZnPM products (days 4-6).
Research assistants weighed each food product before and after feeding to the nearest 0.01 g to determine the net number of grams consumed of each food product. Both research assistants and community health workers assisted mothers with feeding, if needed. The proportion of pearl millet consumed in each food product was then back-calculated via the proportion of pearl millet initially used to prepare the given recipe.
Concurrently with their infants, mothers were also given a sample of each food on the day of its introduction and were asked to rate its acceptability using a 9-point hedonic scale while a research assistant assisted with feeding their child. The hedonic score was based on a 9-point scale (1 = lowest rating, "dislike extremely"; 9 = highest rating, "like extremely") similar to scales used in previous studies of consumer acceptance and assessed four parameters: taste, smell, color, and overall acceptability (18)(19)(20)

ethical approval
Intersystems Biomedica Ethics Committee, Mumbai, India, approved the study protocol (ISBEC/NR-17/KM-JVJ/2014). The nature of the study was explained to the mothers and written informed consent was obtained from the mothers or the caregivers. All participants gave written informed consent.

Data analysis
Two outcome variables in this study were used: the average net number of grams of pearl millet consumed in each food product among children and the mean hedonic score for each food product among mothers. To compare the mean intake of each pearl millet variety as a proportion of the total food product, as well as mean hedonic scores, data were first tested for normality. Because data were not normally distributed, results from the non-parametric Hodges-Lehmann-Sen test were compared with results from two-sample t-tests. Results were similar, thus we report means and t-tests here for comparability with other publications. Anthropometric z-scores [weight-for-age (WAZ), length-for-age (LAZ), and weight-for-length (WLZ)] were calculated using World Health Organization guidelines; underweight, stunting, and wasting were defined as WAZ < −2, LAZ < −2, and WLZ < −2 SDs from the WHO reference standard, respectively (21). SAS version 9.4 (SAS Institute, Cary, NC, USA) was used for all analysis.  Ingredients: pearl millet flour (250 g), powdered sugar (125 g), ghee (83 g), cocoa powder (12.5 g), and baking powder (2.5 g)

Development of Pearl Millet Products
Directions: preheat oven to 120°C (248 F). Sift pearl millet flour, cocoa powder, and baking powder into a bowl and set aside. Cream sugar and ghee. Combine the flour mixture with the creamed ghee and sugar and mix well. Roll out, cut into squares, and bake for 10-12 min. Let cool and serve

FeZnPM cPM
Fe: 5. Ingredients: pearl millet flour (300 g), coarsely ground pearl millet (200 g), water (300 mL), jaggery (200 g), refined wheat flour (50 g), water (192 mL), ghee (20 g), and cardamom powder (1.5 g) Directions: for puran, combine coarsely grouned pearl millet flour and 192 mL water; cook using a pressure cooker. Add jaggery and stir to break up any lumps; add cardamom powder. For poli dough, combine pearl millet flour, wheat flour and 300 mL water and make equal sized balls of dough 3″ in diameter. Flatten each ball into a circular shape (roti) using a rolling pin. On half of the roti, spread an even layer of puran. Place another roti on top and pinch to seal the edges. Roast the puranpoli on both sides. Once cooked through, brush ghee on top and serve warm

FeZnPM cPM
Directions: roast pearl millet in ghee on a medium flame until golden brown. Add carrot and tomato; cook until vegetables are soft. Add hot water and stir continuously, then add spices and green chili paste and cook until water has evaporated and upma has reached a soft consistency. Thin with water as needed. Serve warm.

acceptability: infants and Mothers
Population characteristics of an N = 38 subset of children may be found in Table 2. No statistically significant differences in infants' overall mean consumption of foods developed from ICTP-8203-Fe PM (mean ± SD; 25.3 ± 13.0 g), were found, compared to the conventional pearl millet (21.7 ± 6.9 g) (P = 0.28) ( Table 3). There was no difference in mean grams consumed from either sweet or savory food products in the CPM (P = 0.1) and FeZnPM (P = 0.6) groups. FeZnPM products with the highest acceptability among children included churma laddu (31.5 g), vegetable cutlet (53.2 g), and dhokla (28.1 g), while the least-consumed FeZnPM food products included upma (9.3 g), khichdi (9.8 g), and pav (14.4 g).
Mothers gave higher hedonic scores to foods made with CPM ("Overall" mean hedonic score: 8.22) compared to FeZnPM ("Overall" mean hedonic score: 7.95; P = 0.01) but had no preference or preferred FeZnPM in terms of color, taste, and overall score in at least 50% of recipes (Tables 3-6). In contrast, mothers tended to give higher ratings to CPM in terms of Odor ( Table 5). For specific food items, Mothers gave the highest hedonic ratings to churma laddu, upma, and nankhatai ( Table 3). Bhaji ingredients: coarsely ground pearl millet (150 g), onion (30 g), tomato powder (30 g), green bell pepper (25 g), green chilies (2.5 g), chopped cilantro (2.5 g), pav bhaji spice mix (7.5 g), vegetable oil (30 mL), chili powder (2.5 g), ground turmeric (3 g), salt (7.5 g), and water (490 mL) Directions: pressure cook pearl millet flour for 1-2 whistles on a medium flame. Once cooked, mash the pearl millet. Separately grind two pastes: (1) onion and green pepper, and (2) chilies and garlic. In a pan, heat oil and add onion/pepper paste. Sautée until the onion becomes translucent and then add tomato powder, turmeric, salt, and pav bhaji masala powder. Mix well and fry on medium heat for 2-3 min. Add the cooked pearl millet and mix well with the masala. Add water as needed and stir until desired consistency is reached, then add cilantro. Serve warm with pearl millet pav Ingredients: coarsely ground pearl millet (150 g), pearl millet flour (50 g), green chilies (2.5 g), salt (5 g), ground ginger (3 g), ground turmeric (3 g), cilantro (1.5 g), ground cumin (2.5 g), salt (2.5 g), asafoetida (1.5 g), and water (395 mL), vegetable oil (for frying) Directions: for vada, rinse coarse pearl millet with water and pressure cook. Let cool, then mash until soft. Add 2.5 g salt, ginger, 1.5 g turmeric, cilantro, and 300 water; mix well, form 2″ balls and set aside. In another bowl, mix pearl millet flour with 2.5 g turmeric, cumin, remaining 2.5 g salt, asafoetida, and 1 tsp of hot oil. Add ~95 mL water if necessary to create a thin consistency. Heat oil in a large kadai (pan). Dip each vada ball into the batter and deep fry until golden brown. Allow to cool. Serve with chutney or ketchup     anthropometry Data for sex and age were missing from two participants; therefore, anthropometric z-scores were calculated for the remaining sample of n = 36. On average, mean anthropometric z-scores were below the reference standard for weight-for-length, length-forage, or weight-for-age (

DiscUssiOn
The present study is the first to develop several pearl millet-based complementary foods and to assess the acceptability of these food products among infants living in the urban slums of Mumbai, India. The acceptability of complementary food products was primarily determined by each child's 3-day mean food intake along with mothers' hedonic scores. Complementary food products developed from both varieties of pearl millet were highly accepted by children and their mothers.
There are limited studies on the acceptability of biofortified pearl millet-based complementary foods among young children (22). A recently published randomized controlled trial from our research group studied the effect of consuming biofortified pearl millet on iron status indicators compared to conventional pearl millet in school children from Maharashtra, India (10). These school children regularly consumed pearl millet-based unleavened bread (bhakri) for 6 months. Similar to our findings, no significant differences were observed between the consumption of biofortified and conventional pearl millet, and consumption of bhakri was high, indicating the high acceptance by school children (10). In a hospital-based feeding trial from southern India, the investigators studied the absorption of iron and zinc from biofortified pearl millet-based complementary foods (sheera, upma, and roti) compared to the same food products prepared using conventional pearl millet in young children (22-35 months of age) (16,23). Children consumed an average of ~60 g pearl millet flour per day, again indicating the acceptance of pearl millet-based food products in 22-to 35-month-old children. Acceptability of other biofortified crops such as cassava has also been tested in young children and their caregivers (22). An acceptability study from Kenya compared the sensory acceptability of the consumption of foods prepared using biofortified cassava vs. white cassava in school children (7-12 years of age) and their caregivers (24). This study demonstrated that the biofortified cassava was wellaccepted by the children and their caregivers compared to white cassava (24).
Consistent with our results, high acceptability of cereal and lipid-based complementary foods by toddlers was observed in other feeding studies from Peru (25) and Ghana (26). In our study, though the overall hedonic score responses from the mothers showed that CPM was preferred to FeZnPM, the quantitative data demonstrate that the consumption and acceptability of complementary foods prepared with both FeZnPM and CPM were equally high in this population. Similar hedonic score responses were observed in other studies from Peru (25) and Ghana (26) indicating that mothers' responses can be biased, and direct measure of food consumption by the toddlers are more conclusive evidence of food acceptability, as reported in Bangladesh (27).
We observed variability in consumption volume among children, which may be attributed to the age range included (12-18 months) wherein some children are accustomed to consuming solid food (thereby eating more) while others are just beginning complementary feeding (who may still be breastfeeding, limiting their capacity for the pearl millet). This variability may be minimized by increasing the sample size in future acceptability studies, or excluding children who have already started complementary feeding.
In the preliminary phase of acceptability study, we observed some limitations including mothers' refusal to feed their children in the presence of other caregivers or in a group due to presence of existing socio-cultural myths and taboos. Another important limitation we observed was that some mothers fed their child at home before bringing them to the feeding center as the child were reported to be very hungry. If the child refused to eat the study food product, mothers tended to breastfeed them which may have influenced their child's hunger levels and consumption of the study food products.
In this study, we determined which biofortified pearl millet food products are the most accepted and would, therefore, have a higher likelihood of being consumed as part of the daily diet among young children. For example, churma laddu (described in Table 1) made with FeZnPM variety ICTP-8203 was most accepted by both mothers and infants. This indicates that similar recipes would be ideal candidates for inclusion in the proposed randomized controlled feeding trial (ClinicalTrials. gov ID: NCT02233764; Clinical Trials Registry of India (CTRI), reference number REF/2014/10/007731, CTRI number CTRI/2015/11/006376) to test the efficacy of consuming ironand zinc-biofortified pearl millet like ICTP-8203 in improving iron status, growth, immune function, and cognition among young children.

eThics sTaTeMenT
This study was carried out in accordance with the recommendations of Intersystem Biomedica Ethics Committee (ISBEC) (Ethic